Flow-through paramagnetic particle-based cell separation and paramagnetic particle removal

ABSTRACT

The present disclosure relates to systems and methods for flow-through separation of paramagnetic particle-bound cells in a cell suspension containing both bound and unbound cells as well as systems and methods for removing paramagnetic particles from paramagnetic particle-bound cells or from a cell suspension with unbound cells. It further relates to a flow-through magnetic separation/debeading module and a flow-through spinning membrane debeading module.

PRIORITY CLAIM

The present application claims priority under 35 U.S.C. §119 to U.S.Provisional Patent Application Ser. No. 62/171,787 filed Jun. 5, 2015and U.S. Provisional Patent Application Ser. No. 62/173,702 filed Jun.10, 2015, the contents of which are hereby incorporated by reference intheir entirety.

TECHNICAL FIELD

The present disclosure relates to systems and methods for flow-throughseparation of paramagnetic particle-bound cells in a cell suspensioncontaining both bound and unbound cells as well as systems and methodsfor removing paramagnetic particles from paramagnetic particle-boundcells or from a cell suspension with unbound cells.

BACKGROUND

Particles may be present in the external environment of cells for anynumber of reasons. For example, particles are often coated with agrowth-inducer, which causes responsive cells in a mixed-cell orsingle-cell culture to grow and divide. Particles are also often coatedwith a binding agent, which attaches to a particular type of cell,allowing it to be separated from other types of cells in the samemixed-cell suspension. This separation based on cell type allowsdesirable cells to be separated from undesirable cells.

Although particles are useful for their intended function, the presenceof particles in a cell product may later be detrimental. For instance,the particles themselves may pose a risk of harm to a patient receivingthe cell product for cell therapy. In other cases, the particles mayhamper further growth and division of the cells, or they may need to beremoved so that growth and division slow or to allow the cells todifferentiate.

Various types of particles may be removed from the cell product in anyof a number of ways, but paramagnetic particles are often used becausetheir attraction to a magnet allows both separation of paramagneticparticle-bound cells from unbound cells and paramagnetic particleremoval from paramagnetic particle-bound cells.

Current systems and methods using paramagnetic particles for cellseparation place a cell suspension containing paramagneticparticle-bound cells in a suspension fluid in a chamber, then position amagnet near a chamber wall. Free paramagnetic particles and those boundto cells are attracted to the magnet and, therefore, are held inposition on the inner wall of the chamber adjacent to the magnet. Theremaining suspension fluid, containing any unbound cells, is removed.Then the magnet is moved away from the chamber wall, releasing theparamagnetic particles and any paramagnetic particle-bound cells.

Such a system and method may be used for either positive or negativeselection of paramagnetic particle-bound cells, but it has manydrawbacks in either instance. One drawback is that paramagneticparticles and paramagnetic particle-bound cells may enter the unboundcell product via the unbound cell output fraction because there is notsufficient surface area to accommodate them on the chamber wall, becauseother cells impede them reaching the chamber wall, because they are partof a cell clump that is too large to remain magnetically attached to thechamber wall, or for other reasons. If the unbound cells are to be usedclinically, this contamination is a health hazard. If the unbound cellsare waste, then desirable cells are lost.

Another drawback is that unbound cells may be trapped in layers ofparamagnetic particle-bound cells, again resulting in cells being in thewrong cell output fraction, which may lead to waste or unwantedcontamination.

Layering of cells on the chamber wall presents yet another drawback inthat this layering may cause the cells to clump, interfering with laterprocessing or use, or their access to nutrients. Layering may even causesome cells to be crushed, either destroying desirable cells orintroducing cell lysis contaminants into the cell suspension.

Similar systems and methods are used to remove paramagnetic particlesfrom cells in a process called debeading. In debeading, a cellsuspension is placed in a chamber and a magnet positioned near thechamber wall to attract paramagnetic particles

Such a system and method also has a number of drawbacks including thepossible inclusion of cells with paramagnetic particles in the unboundcell fraction and ultimately the unbound cell product, usually becausethey simply were not attracted to the wall. For instance, because amagnetic field is inversely proportional to the square of the distancefrom the magnet, it falls off rather quickly as one moves away from thechamber wall. If too large of a chamber or too weak of a magnet is used,the chances of paramagnetic particle-bound cells ending up in theunbound cell product is higher.

Systems and methods able to separate or debead paramagneticparticle-bound cells while addressing one or more of these drawbacks areneeded.

SUMMARY

In one aspect, the present disclosure provides a cell processing systemincluding at least one cell suspension module; at least one buffermodule; at least one flow-through magnetic separation/debeading module;at least one non-magnetic output module; and at least one magneticoutput module.

In some variations of this system, it may include at least one returnloop returning upstream of at least one flow-through magneticseparation/debeading module; at least two flow-through magneticseparation/debeading modules in parallel; at least two flow-throughmagnetic separation/debeading modules in series; at least one additionalmodule, or any combinations thereof. The at least one additional modulemay include at least one spinning membrane debeading module; at leasttwo spinning membrane debeading modules in parallel; or at least twospinning membrane debeading modules in series. Any of the spinningmembrane debeading modules may include at least one magnet adjacent orproximate to a cylindrical side-wall.

In a more specific variation, the flow-through magneticseparation/debeading module includes a chamber defined by walls andhaving an x-direction, a y-direction, and a z-direction; an inlet and anoutlet arranged on opposite ends of the chamber in the y-direction; andat least two magnets adjacent or proximate a wall of the chamber andarranged to establish a zero gradient line within the chamber betweenthe inlet and the outlet.

In another more specific variation, which may stand alone or be combinedwith the first more specific variation, the spinning membrane debeadingmodule includes a debeading chamber define partially by a cylindricalside-wall; a porous spinning membrane having an interior and orientedco-axially with the cylindrical side-wall; a sample inlet; a wasteoutput module connected to the interior of the spinning membrane; and acell output module connected to the debeading chamber.

In another aspect, the disclosure provides a flow-through magneticseparation/debeading module including a chamber defined by walls andhaving an x-direction, a y-direction, and a z-direction; an inlet and anoutlet arranged on opposite ends of the chamber in the y-direction; andat least two magnets adjacent or proximate a wall of the chamber andarranged to establish a zero gradient line within the chamber betweenthe inlet and the outlet.

In some variations of this module, it includes it least two inlets andat least two outlets;

at least three magnets adjacent or proximate a wall of the chamber andarranged to establish at least two zero gradient lines within thechamber between the inlet and the outlet; at least four magnets arrangedin two arrays on opposite sides of the chamber in the z-direction; atleast four magnets arranged in two arrays on opposite sides of thechamber in the z-direction and cross-oriented in the x-y plane from nearone inlet to near one outlet on the opposite side of the chamber in thez-direction; a sub-membrane injection ports adjacent a wall of thechamber also adjacent at least two magnets and a membrane adjacent thesub-membrane; or any combinations thereof.

In another aspect, the disclosure provides a spinning membrane debeadingmodule including a debeading chamber define partially by a cylindricalside-wall; a porous spinning membrane having an interior and orientedco-axially with the cylindrical side-wall; a sample inlet; a wasteoutput module connected to the interior of the spinning membrane; a celloutput module connected to the debeading chamber; and at least onemagnet adjacent or proximate to the cylindrical side-wall.

In some variations of this module, it may include a reagent module, havea pore size greater than that of a particle to be debeaded and less thanthat of a cell to be debeaded, or both.

In yet another aspect, the disclosure provides a method of flow-throughcell processing by flowing a cell suspension comprising paramagneticparticle-bound cells through a flow-through magneticseparation/debeading module to produce an unbound cell product. Theparamagnetic particle-bound cells continue to move in the flow-throughmagnetic separation/debeading module through the flowing step. Theflow-through magnetic separation/debeading module includes a flowchamber defined by walls through which the cell suspension flows and atleast two magnets arranged adjacent or proximate at least one wall.

In some variations of this method, the cell suspension is flowedlaminarly through the flow-through magnetic separation/debeading module;the cell suspension further includes unbound cells and flowing the cellsuspension through the flow-through magnetic separation/debeading moduleseparates the paramagnetic particle-bound cells and the unbound cells;the cell suspension further includes free paramagnetic particles andflowing the cell suspension through the flow-through magneticseparation/debeading module separates the free paramagnetic particlesand the unbound cells, or any combinations thereof.

The method may also include flowing the separated unbound cells throughthe flow-through magnetic separation/debeading module a second orsubsequent time using a return loop; flowing the separated paramagneticparticle-bound cells through the flow-through magneticseparation/debeading module a second or subsequent time using a returnloop; debeading the paramagnetic particle-bound cells in theflow-through magnetic separation/debeading module during the second orsubsequent time to produce paramagnetic particles and debeaded, unboundcells; flowing the produced paramagnetic particles and debeaded, unboundcells through the flow-through magnetic separation/debeading module athird or subsequent time to separate the paramagnetic particles and thedebeaded, unbound cells, or any combinations thereof.

In another variation, combinable with all others, the magnets areoriented to establish one zero gradient line that crosses the directionof flow, such that paramagnetic-particle bound cells are pulled to thezero gradient line in one direction only, but are not affected bymagnetic forces from the magnets in two other directions.

In another variation, combinable with all others, the chamber furtherincludes a magnetic inlet through which any paramagnetic particles enterthe flow chamber; a non-magnetic inlet; a magnetic outlet opposite thenon-magnetic inlet; and a non-magnetic outlet opposite the magneticinlet, wherein the zero gradient line directs all paramagnetic particlesand any paramagnetic particle-bound cells to the magnetic outlet.

The cell suspension may further include unbound cells and thenon-magnetic inlet may be larger than the magnetic inlet while thenon-magnetic outlet is larger than magnetic outlet, so that fluidflowing from the non-magnetic inlet crosses over to the non-magneticoutlet, preventing any unbound cells from flowing into the magneticoutlet.

Alternatively, the cell suspension may further include unbound cells,and the non-magnetic inlet and magnetic inlet may be substantially thesame size or the non-magnetic outlet and magnetic outlet may besubstantially the same size, or both, and the respective flow rates ofthe fluid entering the inlets, the respective flow rates of the fluidexiting the outlets, or both may be adjusted such that fluid flowingfrom the non-magnetic inlet crosses over to the non-magnetic outlet,preventing any unbound cells from flowing into the magnetic outlet.

In another variation, combinable with all others, the method may includeflowing the paramagnetic particle-bound cells through a spinningmembrane debeading module to produce the unbound cell product. Thespinning membrane debeading module may include a cylindrical debeadingchamber through which the paramagnetic particle-bound cells flow, thechamber defined in part by a cylindrical side-wall and containing aco-axial spinning membrane; and at least one magnet arranged adjacent orproximate the cylindrical side-wall to establish at least one zerogradient line within the cylindrical debeading chamber.

In another aspect, the disclosure provides a method of manufacturing acell therapy composition by contacting a cell population withparamagnetic particles coated with one or more agents which assist inexpanding one or more cell types within the cell population; introducingnucleic acid into cells within the cell population; expanding cellswithin the cell population; debeading the cell population according toany of the above systems or methods or any other system or methoddescribed herein; and formulating the cell population for cell therapy.

In some variations, the one or more agents which assist in expanding oneor more cell types may include anti-CD3 antibody or antigen bindingfragment thereof, anti-CD28 antibody or antigen binding fragmentthereof, and combinations thereof; the nucleic acid may be introduced bylentivirus or mRNA transduction; the cell therapy may be a chimericantigen receptor T cell therapy; the cell therapy is an anti-CD19chimeric antigen receptor T cell therapy; or any combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete understanding of the present disclosure and advantagesthereof may be acquired by referring to the following description takenin conjunction with the accompanying drawings, which are not to scaleand in which like numerals refer to like features:

FIG. 1A is a schematic diagram of a cell processing system with aflow-through magnetic separation/debeading module;

FIG. 1B is a schematic diagram of a cell processing system with aplurality of flow-through magnetic separation/debeading modules inparallel;

FIG. 1C is a schematic diagram of a cell processing system with aplurality of flow-through magnetic separation/debeading modules inseries;

FIG. 1D is a schematic diagram of a cell processing system with a returnloop;

FIG. 1E is a schematic diagram of a cell processing system with cellsuspension and buffer modules separately connected to the flow-throughmagnetic separation/debeading module;

FIG. 1F is a schematic diagram of a cell processing system including aspinning membrane debeading module;

FIG. 1G is a schematic diagram of a cell processing system includingmultiple flow-through magnetic separation/debeading modules and multiplespinning membrane debeading modules;

FIG. 1H is another schematic diagram of a cell processing system with aflow-through magnetic separation/debeading module;

FIG. 2A is a transverse cross-sectional schematic diagram of aflow-through magnetic separation/debeading module in an x-orientedmagnet configuration;

FIG. 2B is a semi-transparent, three-dimensional schematic diagram ofthe flow-through magnetic separation/debeading module of FIG. 2A;

FIG. 3 is a side longitudinal cross-sectional schematic diagram of aflow-through magnetic separation/debeading module with a membrane andsub-membrane fluid injection ports;

FIG. 4A is a transverse cross-sectional schematic diagram of aflow-through magnetic separation/debeading module in a zero-gradientconfiguration;

FIG. 4B is a semi-transparent, three-dimensional schematic diagram ofthe flow-through magnetic separation/debeading module of FIG. 4A;

FIG. 4C is a top longitudinal cross-sectional schematic diagram of aflow-through magnetic separation/debeading module in a zero-gradientconfiguration to create a zero gradient filter;

FIG. 4D is a top longitudinal cross-sectional schematic diagram of aflow-through magnetic separation/debeading module in a multiplezero-gradient configuration to create a multiple zero-gradient filter;

FIG. 5A is a longitudinal cross-sectional schematic diagram of aspinning membrane debeading module;

FIG. 5B is a transverse cross-sectional schematic diagram of a spinningmembrane debeading module with a first magnet configuration;

FIG. 5C is a transverse cross-sectional schematic diagram of a spinningmembrane debeading module with a second magnet configuration;

FIG. 6 is diagram of fluidic force and magnetic force on a cell;

FIGS. 7A, 7B, and 7C are diagrams of the flow-through magneticseparation/debeading module of FIG. 2B with cells present duringmagnetic separation;

FIGS. 8A and 8B are diagrams of the flow-through magneticseparation/debeading module of FIG. 2B with cells present duringmagnetic debeading;

FIG. 9 is a graph comparing the results of debeading using theflow-through magnetic separation/debeading module of FIGS. 2A and 2B andthe results of debeading using a conventional stop-flow module (boxesrepresent quartiles and the median);

FIG. 10A is a top longitudinal x-y cross-sectional schematic diagram ofa flow-through magnetic separation/debeading module with paramagneticparticle-bound cells present near the module inlet (left) and near themodule outlet (right);

FIG. 10B is a side longitudinal cross-sectional schematic diagram of aflow-through magnetic separation/debeading module with paramagneticparticle-bound cells present near the module inlet (left) and near themodule outlet (right);

FIG. 11 is a diagram of the flow-through magnetic separation/debeadingmodule similar to that of FIG. 4A during magnetic separation ofparamagnetic particle-bound cells and unbound cells;

FIG. 12 is a diagram of the flow-through magnetic separation/debeadingmodule of FIG. 4C with cells present during magnetic separation ofparamagnetic particle-bound cells and unbound cells;

FIG. 13 is a diagram of the flow-through magnetic separation/debeadingmodule of FIG. 4C with cells present during magnetic separation ofparamagnetic particles from debeaded, unbound cells;

FIG. 14 is a diagram of the spinning membrane debeading module of FIG. 5with cells present during debeading;

FIG. 15 is a graph of the harvest yields for a non flow-throughdebeading process and a flow-through debeading process as a function ifinput cell number; and

FIG. 16 is a binned graph of the harvest yields for a non flow-throughdebeading process and a flow-through debeading process as a function ofinput cell number.

DETAILED DESCRIPTION

The present disclosure relates to systems and methods for flow-throughseparation, debeading, paramagnetic particle separation, or anycombination thereof of paramagnetic particle-bound cells or unboundcells in the presence of paramagnetic particles, in a cell suspension.The systems and methods use a flow-through magnetic separation/debeadingmodule, a spinning membrane debeading module, or both. Although thesystems and methods described herein may be used to remove paramagneticparticles from any type of cell, they are particularly well-adapted foruse in removing paramagnetic particles from cells to be used in celltherapy. In addition, although some portions of the description focus onpositive selection of paramagnetic particle-bound cells, as debeading istypically only performed during positive selection methods, the systemsand methods may also be used for negative selection. When used fornegative selection, typically any debeading modules and steps will beeliminated.

Separation and Debeading Systems and Modules

FIG. 1A is a schematic diagram of a cell processing system 2 forflow-through separation, debeading, paramagnetic particle separation, orany combination thereof of paramagnetic particle-bound cells in a cellsuspension. System 2 includes cell suspension module 4, buffer module 6,flow-through magnetic separation/debeading module 8, non-magnetic outputmodule 10, and magnetic output module 12. System 2 may optionally alsoinclude at least one additional module 16, at least one return loop 14,or both.

System 2 additionally may include fluid conduits, such as tubes orhoses, connectors, valves, switches, clamps, weld sites, housings,motors, pumps, other mechanical mechanisms, circuitry, monitoringdevices, and control devices. System 2 may further include a computerprogrammed to control system 2 or any component thereof to perform aflow-through separation process, a flow-through debeading process, aflow-through paramagnetic particle separation process, or anycombination thereof. System 2 may have a static configuration, or it mayhave an adaptable configuration.

One adaptable configuration may allow the exchange of modules or theinsertion of additional modules. Another adaptable configuration mayhave an unchangeable set of modules, but may allow changes in fluidrouting to at least one of the modules. Other adaptable configurationsmay allow both exchange and addition of modules as well as changes influid routing. Components of system 2 may facilitate the adaptableconfiguration. For instance, a programmed computer in system 2 maydetect or use information regarding which modules are present or it maycontrol fluid routing. In addition, system 2 may be have housings orfluid conduits with accompanying connectors, valves, clamps, or switchesthat allow removal or insertion of different modules in the samelocation. Modules or other components may contain identificationelements, such as bar codes or radio frequency identification (RFID)chips, to allow their presence or absence to be automatically detected.Modules or other components may also contain one or more indicationelements, which may ensure compliance with good manufacturing practicesand other safety regulations. For instance, temperature sensitiveindication elements may indicate that a module or other component hasbeen heat sterilized or not subjected to a temperature that maycompromise its integrity or effectiveness. Indication elements may alsoclearly identify used modules or other components. Indication elementsmay also be automatically detected by system 2, helping to minimizehuman error.

Components of system 2 not needed for a particular process may beabsent, unconnected, or closed. For instance, a single output module maybe present rather than a separate non-magnetic output module 10 andmagnetic output module 12, as shown in FIG. 1H. In addition, also asillustrated in FIG. 1H, the components of system 2 may have fluidconduits with different routes and connections than as shown in FIG. 1A,depending on the configuration of valves, clamps, weld sites, switches,and connectors.

Cell suspension module 4 contains cells to be separated or debeadedsuspended in a suspension fluid. If the cells are to be separated, thentypically the cell suspension contains both paramagnetic particle-boundcells and unbound cells. The paramagnetic particle-bound cells may bethe desirable cells, in which case positive selection for paramagneticparticle-bound cells will occur in system 2, or the paramagneticparticle-bound cells may be undesirable, in which case negativeselection for the paramagnetic particle-bound cells will occur.

If the cells are to be debeaded, or if paramagnetic particles are to beseparated from the cells, then the paramagnetic particle-bound cells aredesirable cells. They may have previously been separated fromundesirable cells using system 2 or another system. In instances wherethe presence of undesirable cells is not problematic or where there areno undesirable cells to separate, the cells to be debeaded may not havepreviously undergone a separation process.

The cells may be obtained directly from a biological sample, such asblood, or from a cell culture.

The suspension fluid may be any fluid able to support viability of thecells throughout the separation, debeading, or particle removal process.For instance, it may be a culture medium, a freezing agent, such as aDMSO-containing fluid, another fluid with a set or controlled pH, oranother fluid with nutrients. It may also be a buffer, which may be thesame as or different from the buffer in buffer module 6. The suspensionfluid may have a different viscosity than the buffer. It may also have adifferent viscosity than the medium in which the cells enter system 2,which may be very dense, such as high density Ficoll.

The buffer in buffer module 6 may be any fluid that may be combined withthe suspension fluid while allowing the suspension fluid to continue tosupport viability of the cells. For instance, the buffer may have a setor controlled pH. The buffer may include one or more cell-compatiblesalts. Although buffer is provided separately in buffer module 6, oncethe buffer mixes with the cell suspension, it is considered to be partof the suspension fluid.

The suspension fluid or buffer may contain antimicrobial agents, buttypically will not if the cells will later be provided to a patientunless system 2 removes these agents, such as via a spinning membranedebeading module or another module, or unless they are removed later byan additional process, module, or system.

The paramagnetic particles may be formed from any paramagnetic and/ormagnetizable material, such as a metal or metal alloy. Typically theparamagnetic material is not toxic to the cells or to any patient whowill later receive the cells, or it is coated to avoid toxicity. Theparamagnetic material may be selected to achieve a high magneticsaturation flux (m_(s)). In general, which paramagnetic materials aresuitable is influenced by the magnets used in system 2 and theconfiguration of modules using the magnets, as these elements influencedriving the paramagnetic material to magnetic saturation.

The paramagnetic particles may be coated with a binding agent, such as agrowth agent, a receptor or ligand, an antigen, an antibody, or anybinding fragments or chimeric variants thereof, such as a chimericantigen receptor ligand. The binding agents may be reversible in someinstances, allowing detachment of the paramagnetic particlesspontaneously or using a particular chemical agent. The binding agentsmay also include a photo-cleavable linker, in which case system 2 mayinclude a light source, particularly a high power light source, as amodule or as part of another module to allow photo-cleavage of thelinker and separation of the cell and paramagnetic particle. In someinstances, the coating may interact with the cells. In other instances,the coating may interact with at least one unwanted constituent of thecell suspension that is to be removed. The unwanted constituent may beactive or inactive and may have previously served a useful function withrespect to the cells or the cell suspension fluid. Example unwantedconstituents include antibodies, growth factors, other proteins, andpolymers.

Different types of paramagnetic particles, such as particles withdifferent binding agents or formed from different magnetic materials maybe present in some cell suspensions, allowing for complex separations oriterative removal of binding agents. Additional, non-paramagneticparticles, which may also be coated with any binding agent, may also bebound to cells.

Other particles that are not paramagnetic may also be present in thecell suspension and may be coated with anything used to coat theparamagnetic particles.

Modules may be formed from or lined with any biologically compatiblematerial such as cell storage bags. Fluid conduits and any othercomponent of system 2 that contacts the cell suspension or buffer mayalso be formed from or lined with any biologically compatible material.

Components of system 2 that will contact the cell suspension or buffermay be sterile prior to contact with the cell suspension.

Components of system 2 may be disposable. Components that contact thecell suspension, in particular, may be disposable to avoid contaminationand sterility concerns.

FIG. 2A is a transverse cross-sectional schematic diagram offlow-through magnetic separation/debeading module 8 in an x-orientedmagnet configuration, while FIG. 2B is a semi-transparent,three-dimensional schematic diagram of the same magnetic separationmodule 8 in the same configuration. Flow-through magneticseparation/debeading module 8 includes a flow chamber 50, defined bywalls 52. External dipole magnets 54 create magnetic force lines 56.Magnets 54 are housed on movable platform 60. Flow-through magneticseparation/debeading module 8 further includes inlet 62 and outlet 64through which a cell suspension may be flowed in the y direction throughmodule 8.

Although FIGS. 2A and 2B depict one array of magnets 54, flow-throughmagnetic separation/debeading module 8 may have two or more arrays, asshown in FIG. 4A, and may have more than two magnets in an array. Inaddition, magnets 54 may be in a permanent position, in which caseseparating/debeading module 8 may lack movable platform 60 or may have amovable flow chamber 50. Furthermore, although magnets 54 are shown inan x-oriented configuration, they can be at any angle in the x-y plane,including a y-orientation or an x-y cross-orientation.

Inlet 62 and outlet 64 may have any configuration sufficient toestablish laminar flow of the cell suspension through chamber 50. Inletgeometry, outlet geometry, and flow rate all influence the flow of thecell suspension through chamber 50. Turbulent flow may be acceptable insome instances.

Walls 52 may be rigid structures, or they may be flexible. For instance,they may be the walls of a cell storage bag or other similar component.When walls 52 are flexible, the dimension of chamber 50 in the zdirection may vary depending on the flow rate of the cell suspensionthrough chamber 50.

The dimension of chamber 50 in the z direction may be between 5 μm and100 μm, between 5 μm and 500 μm, or between 5 μm and 1000 μm, between 5μm and 1 cm, or generally 100 μm, 500 μm, 1000 μm, or 1 cm or less. Thedimensions in both the x, y, and z directions may be limited to achievefluidic forces that are sufficiently high to move cells or paramagneticparticles through chamber 50.

Magnets 54 may have a high magnetic field strength. For instance, theymay contain rare earth metals, such as neodymium or samarium alloyedwith another metal, such as cobalt. Magnets 54 may be dipole magnets asdepicted, or they may be other types of magnets, such as quadrapolemagnets. Magnets 54 may have an adjustable magnetic field strength. Forexample, they may be electromagnets. Magnets 54 may be arranged tomaximize magnetic attraction for magnets on the same side of chamber 50,to maximize magnetic repulsion for magnets on opposite sides of chamber50, or both. Although FIG. 2A depicts a particular magnet configuration,configurations in which magnet polarity is opposite or concordant may beused depending upon the effect to be achieved.

FIG. 3 is a side longitudinal cross-sectional schematic diagram of aflow-through magnetic separation/debeading module 8 with membrane 70located above sub-membrane fluid injection ports 72 and magnets 54.Fluid from buffer module 6 or another fluid module may be introducedthrough fluid injection ports 72 to help debead cells located nearmembrane 70.

Movable platform 60 may be movable in the z direction allowing themovement of magnets 54 in the z direction from a position adjacent tochamber 50 as shown in FIGS. 2A and 2B, or proximate chamber 50 (notshown), to a position distant from chamber 50 (as shown in FIG. 7C). Forexample, the position distant from chamber 50 may be at least 1 cm fromthe nearest wall 52. The position distant is sufficient to prevent anysubstantial influence of magnets 54, via their magnetic fields, on anyparamagnetic particle in chamber 50. The position distance may besubstantially less if a magnetically insulating material is insertedbetween magnets 54 and chamber 50. If the magnets 54 have an adjustablemagnetic field strength, rather than being moved, they may simply beadjusted to a lower magnetic field strength or zero magnetic fieldstrength to avoid any substantial influence on any paramagneticparticles in chamber 50.

Particularly when using a zero-gradient configuration, module 8 may havea top array of magnets 54 and a bottom array of magnets 54, as depictedin FIGS. 4A and 4B. Movable platform 60 may be rotatable in the x-yplane, or magnets 54 may be permanently oriented such that magnets 54are in an x-y cross-oriented configuration, such as that depicted in thetop longitudinal cross-sectional schematic diagram of a flow-throughmagnetic separation/debeading module 8 of FIG. 4C. For use in azero-gradient configuration, flow-through magnetic separation/debeadingmodule 8 may have two inlets 62, a non-magnetic inlet 62 a and amagnetic inlet 62 b as well as two outlets 64, a magnetic outlet 64 aand a non-magnetic outlet 64 b. In this instance, the zero gradient line58 in an x-y cross-oriented direction forms a zero gradient filter whenmodule 8 is in use. Inclusion of additional magnets 54 may provide twozero gradient lines, 58 a and 58 b in the same module 8, as illustratedin FIG. 4D, allowing the separation of different paramagnetic particlesinto different outlets 64 a and 64 b, or providing a back-up filter.

Zero gradient line 58 may be a zero gradient band, having a dimension inthe x direction, if magnets 54 are spaced sufficiently apart from oneanother rather than being adjacent as depicted in FIGS. 2A and 2B.

Magnets for use with a flow-through magnetic separation/debeading modulemay be located external to the chamber that the cell suspension flowsthrough, or internal to the chamber. If the magnets are internal, theymay be coated with a biocompatible material. Particularly if the magnetsare internal, they may be disposable.

FIG. 1B is a schematic diagram of a cell processing system 2, whichincludes a plurality of flow-through magnetic separation/debeadingmodules 8 a, 8 b and 8 c in parallel. Although only three flow-throughmagnetic separation/debeading modules 8 are illustrated, the pluralitymay be any number greater than two. When flow-through magneticseparation/debeading modules 8 are in parallel, the modules willtypically be of the same type and in the same configuration so that thesame function is performed by each. Parallel flow-through magneticseparation/debeading modules 8 may be particularly useful for rapid cellsuspension processing or management of fluid volume when combined withadditional modules. In addition, placing flow-through magneticseparation/debeading modules 8 in parallel provides flexibility incontrolling fluid flow, as the modules need to all be used at the sametime.

FIG. 1C is a schematic diagram of a cell processing system 2, whichincludes a plurality of flow-through magnetic separation/debeadingmodules 8 a, 8 b and 8 c in series. Although only three flow-throughmagnetic separation/debeading modules 8 are illustrated, the pluralitymay be any number greater than two. When flow-through magneticseparation/debeading modules 8 are in series, they may be of the sametype and configuration so that the same function is performed by each,but, typically, they will be of different types and configurations sothat different functions are performed by each. For instance module 8 amay separate paramagnetic particle-bound cells and unbound cells, module8 b may debead paramagnetic particle-bound cells, and module 8 c maydebead paramagnetic particle-bound cells under greater magnetic fieldgradients.

FIG. 1D is a schematic diagram of a cell processing system 2 in whichreturn loop 14 directs paramagnetic particle-bound cells back throughflow-through magnetic separation/debeading module 8. Such a system maybe used to achieve better separation of paramagnetic particle-boundcells and unbound cells, or better debeading or separation of unboundcells and paramagnetic particles as compared to a similar system with noreturn loop 14. Fluid may be directed to return loop 14 or to magneticoutput module 12 by a valve or switch.

FIG. 1E is a schematic diagram of a cell processing system 2 in whichcell suspension module 4 and buffer module 6 are separately connected toflow-through magnetic separation/debeading module 8. Such a system maybe particularly useful when flow-through magnetic separation/debeadingmodule 8 has two inlets 62 and two outlets 64 and magnets 54 in an x-ycross-oriented configuration as shown in and described with respect toFIG. 5.

FIG. 1F is a schematic diagram of a cell processing system 2 with aspinning membrane debeading module 18 located downstream of flow-throughmagnetic separation/debeading module 8. Spinning membrane debeadingmodule 18 is connected to waste output module 20 and cell output module22. In this system 2, flow-through magnetic separation/debeading module8 separates paramagnetic particle-bound cells and unbound cells, while aspinning membrane debeading module 18 conducts all debeading, orflow-through magnetic separation/debeading module 8 may conductdebeading as well. Reagent module 24 may optionally be present if areagent, such as a chemical agent, is added to the suspension fluid inspinning membrane debeading module 18. Although one spinning membranedebeading module 18 is illustrated in FIG. 1F, system 2 may include aplurality of modules 18 in series or in parallel. When modules 18 are inseries, a chemical agent may only be added to the last module 18 tominimize cell exposure to the chemical agent.

The reagent in reagent module 24 may be any chemical agent that weakensthe bond between a particle and a cell. The particle may be theparamagnetic particle, or it may be a non-paramagnetic particle.

FIG. 5A is a longitudinal cross-sectional schematic diagram of aspinning membrane debeading module 18. This module 18 includes sampleinlet 80, which allows a cell suspension fluid to flow into cylindricaldebeading chamber 82, which is defined in party by cylindrical side-wall84 and contains co-axially oriented cylindrical spinning membrane 86.Wall 84 is lined on the exterior with magnets 88. Debeading chamber 18allows fluid that has passed through spinning membrane 86 to exit viawaste output module 20, while the remaining fluid and cells exit throughcell output module 22. Spinning membrane 86 has an average pore sizesmaller than the average diameter of the cells, but larger than anynon-paramagnetic particle to be removed by debeading. The average poresize may also be larger than any paramagnetic particle to be removed,allowing removal of these paramagnetic particles by the spinningmembrane either as a primary particle removal method, or as a back-up tomagnetic removal.

Magnets 88 may substantially surround wall 84 as shown in FIG. 5B, orthey may be spaced at intervals along wall 84, as shown in FIG. 5C.Magnets 88 may be mounted on a movable platform to allow them to bemoved from a position adjacent to wall 88, as shown in FIGS. 5A-5C, orproximate walls 88 (not shown) to a position distant from wall 84. Forexample, the position distant may be at least 1 cm from wall 84. Thismovement to a position distant prevents magnets 88, via their magneticfield, from having a substantial influence on any paramagnetic particlesin chamber 82. If a magnetically insulating material is inserted betweenmagnets 88 and chamber 82, the position distant may be less than if themagnetically insulating material were not present. If the magnets 88have an adjustable magnetic field strength, rather than being moved,they may simply be adjusted to a lower magnetic field strength or zeromagnetic field strength to avoid an substantial influence on anyparamagnetic particles in chamber 82.

Although multiple magnets 88 are shown in FIG. 5, it is possible to haveonly a single magnet 88.

Magnets 88 may have a high magnetic field strength. For instance, theymay contain rare earth metals, such as neodymium or samarium alloyedwith another metal, such as cobalt. Magnets 88 may be dipole magnets,quadrapole magnets, or any other type of magnets. Magnets 88 may have anadjustable magnetic field strength, for example, they may beelectromagnets. Magnets 88 may be arranged to maximize magneticattraction, for instance in a wrapped configuration.

Magnets for use with a spinning membrane module may be located externalto the chamber that the cell suspension flows through, or internal tothe chamber. If the magnets are internal, they may be coated with abiocompatible material. Particularly if the magnets are internal, theymay be disposable.

Example spinning membranes suitable for use in modules disclosed hereininclude the 4-μm track-etched polycarbonate spinning membrane used inthe LOVO® cell processing system (Fresenius Kabi, Fenwal, Lake Zurich,Ill.), and the spinning membrane used in the ISOLEX® magnetic cellseparation systems (Baxter, Deerfield, Ill.).

Elements from FIGS. 1A-1F, including flow-through magneticseparation/debeading modules 8 as described in FIGS. 2-4 or magneticspinning membrane debeading modules 18 as described in FIG. 5 may becombined with one another in cell processing system 2 depending on thespecific cell processing to be performed. The elements may be combinedas depicted, or in other reasonable variations. For instance, a reagentmodule 24 may be included in a system otherwise a depicted in FIG. 1A sothat a chemical agent may be added to suspension fluid in flow-throughmagnetic separation/debeading module 8 when it is used for debeading.Modules, including additional buffer modules or output modules, may bearranged and used to ensure proper fluid volumes and flow rates,particularly in modules 8 and 18.

One example cell processing system 2 combining multiple modules andloops is illustrated in FIG. 1G. This system includes cell suspensionmodule 4 and buffer module 6 a connected to first flow-through magneticseparation/debeading module 8 a, which has non-magnetic output module 10a and magnetic output module 12 a. Magnetic output module 12 a isconnected to second, in-series flow-through magneticseparation/debeading module 8 b, which is also connected to buffermodule 6 b and has non-magnetic output module 10 b and magnetic outputmodule 12, and return loop 14 a. Return loop 14 a leads back to module 8b. Magnetic output module 12 b leads to first spinning membranedebeading module 18 a, which has waste output module 20 a and celloutput module 22 a. Cell output module 22 a leads to second, in-seriesspinning membrane debeading module 18 b, which is also connected toreagent module 24 as well as waste output module 20 b, return loop 14 b,and cell output module 22 b. Return loop 14 b leads back to second,in-series, flow-through magnetic separation/debeading module 8 b.

Another example cell processing system 2, having various additionalmodules with specific fluid conduits, is shown in FIG. 1H. Cellsuspension module 4 and satellite module 30, which may be empty or maycontain buffer, are connected to flow-through magneticseparation/debeading module 8. Flow-through magneticseparation/debeading module 8 is separately connected to buffer module 6and reservoir module 26. Reservoir module 26 is further connected torecovery module 28. Many connections are made using spike tubing 32. Thesystem further contains, along various fluid conduits, roller clamps 34,weld sites 36, a slide clamp 38, and pinch clamps 40.

Cell processing system 2 may contain a variety of additional modules 16,such as magnetic columns, other physical separation modules, cellwashing modules, cell concentration modules, and media exchange modules.

Cell Separation and Debeading Methods

System 2 may be used to separate paramagnetic particle-bound cells andunbound cells, to debead magnetic-particle bound cells, to separateparamagnetic particles and unbound cells, or any combination thereof, ina flow-through process. In a flow-through process, all cells continue tomove while in flow-through magnetic separation/debeading module 8. None,no more than 0.01%, or no more than 0.05%, or no more than 1% ofparamagnetic particle-bound cells passing through module 8 stop alongwalls 52.

Flow-Through Magnetic Separation/Debeading Processes

In a flow-through process using the system of FIG. 1A, flow-throughmagnetic separation/debeading module 8 may optionally be primed byflowing buffer from buffer module 6 through it to either non-magneticoutput module 10 or magnetic output module 12, or another output oradditional module 16. A cell suspension containing paramagneticparticle-bound cells is flowed through module 8.

If system 2 is configured for separation, the cell suspension isdirected to non-magnetic output module 10. Module 8 may periodically beconfigured to not attract paramagnetic particles while buffer frombuffer 6 flows through it to magnetic output module 12. In order toensure better separation of cells, and high purity of the magnetic ornon-magnetic cell products, the cell suspension may be directed througha loop 14 for a second or subsequent passage through module 8.Paramagnetic particle-bound cells may be directed to an additionalcomponent 16, such as a flow-through magnetic separation/debeadingmodule 8 configured for debeading or to a spinning membrane debeadingmodule 18.

If system 2 is configured for debeading, after flowing through module 8,the cell suspension is directed to non-magnetic output module 10.Alternatively, the cell suspension may be directed through loop 14 for asecond or subsequent passage through module 8 prior to direction tonon-magnetic output module 10.

Processes using flow-through magnetic separation/debeading module 8subject each paramagnetic particle-bound cell 100 with at least onebound paramagnetic particle 102 to a fluidic (shear or drag) force 104.Each cell 100 is also subjected to magnetic force 106, as shown in FIG.6. Fluidic force 104 and magnetic force 106 may be combined in such away that paramagnetic particle 102 remains bound to paramagneticparticle-bound cell 100, allowing cell 100 to be separated form unboundcells. Fluidic force 104 and magnetic force 106 may also be combined insuch a way as to cause paramagnetic particle 102 to detach fromparamagnetic particle-bound cell, allowing debeading of cell 100.Secondary forces, such a diffusion and gravity, also act uponparamagnetic particle 102, but their effects on debeading are typicallymuch less than fluidic force and magnetic force and are often ignoredwhen calculating the proper flow rate through a module.

System 2 may generally be configured so that, as quickly as possible,desirable cells are no longer subject to passage through modules,reducing trauma to the cells. For instance, only the magnetic outputmodule 12 may contain a return loop to flow-through magneticseparation/debeading module 8 in a debeading configuration, allowingmore easily debeaded cells to pass through module 8 fewer times thanthose with more recalcitrantly bound paramagnetic particles.

Flow-through module 8 may be laminar or turbulent. Magnetic force 106 isinfluenced by the saturation flux (m_(s)) of paramagnetic particle 102.Magnetic force 106 is also influenced by the strength of the magneticfield 56 to which paramagnetic particle 102 is subjected, which isinfluenced by the strength of magnets 54 as well as depth of chamber 50in the z direction and cell 100's location within that chamber. Magneticforce 106 is further influenced by the magnetic field gradient to whichparamagnetic particle 102 is subjected as it moves through chamber 50,which is influenced by the strength and placement of magnets 54 as wellas paramagnetic particle 102's location with respect to magnets 54.

In addition, fluidic forces are affected by the fluid flow velocity,which is typically highest in the center of the chamber and zero at thewalls, meaning that cells attached to the walls may not experiencesufficient fluidic force to detach them from their paramagnetic particleand that cells along the wall may be largely stationary and may shielddownstream cells from fluidic forces. This results in loss of desirablecells and is avoided by continuous flow of the cell suspension throughchamber 50. This loss may be avoided by a flow-through approach, inwhich cells are not rendered stationary by magnetic force. It may alsobe avoided by modules in which velocity is not zero or near zero at thewalls, such as plug flow modules where fluid flow velocity is uniformacross the chamber.

Flow-Through Separation Process

A flow-through separation process may be conducted using flow-throughmagnetic separation/debeading module 8 in a configuration as shown inFIGS. 2A and 2B. Flow rate is such that fluidic force 104 and magneticforce 106 allow paramagnetic particle 102 to remain bound to cell 100.Flow rate is also such that cell 100 is not lysed by fluidic forces.

FIG. 7A shows module 8 of FIG. 2B when paramagnetic particle-bound cell100 and unbound cell 110 have recently entered chamber 50. In FIG. 7B,cell 100 has stopped, while unbound cell 110 has continued on at itoriginal velocity. In FIG. 7C, the magnets have been moved away and cell100 continues to move, but unbound cell 110 has exited chamber 50.

Suspension fluid exiting chamber 50 while magnets 54 are adjacent to orproximate chamber 50 enters non-magnetic output module 10. Periodically,cell suspension flow from cell suspension module 4 is stopped and bufferis flowed into chamber 50 from buffer module 6 while magnets 54 aremoved away from chamber 50 to allow paramagnetic particle-bound cells100 to be flushed into magnetic output module 12 by the buffer.

This flow-through separation process, particularly when repeated toallow multiple passages of cells through module 8, may remove at least80%, at least 90%, at least 95%, or at least 99% of paramagneticparticle-bound cells 100 from the cell suspension prior to its entryinto non-magnetic output module 10. Efficiency may be lower for a singlepass process, in which cells pass through module 8 only once. Forinstance, in a single pass process, module 8 may remove at least 25% orat least 50% of paramagnetic particle-bound cells 100 from the cellsuspension prior to its entry into non-magnetic output module 10. Singleor multiple passes of either the non-magnetic output or the magneticoutput through flow-through magnetic separation/debeading module 8 mayresult in paramagnetic particle-bound cell product with no more than 1%unbound cells 110 and containing at least 99% of the paramagneticparticle-bound cells 100 found in the cell suspension prior to theflow-through separation process, an unbound cell product with no morethan 1% paramagnetic particle-bound cells 100 and containing at least99% of the unbound cells 110 found in the cell suspension prior to theflow-through process, or both.

Flow-Through Debeading Process

A flow-through debeading process may also be conducted usingflow-through magnetic separation/debeading module 8 in a configurationas shown in FIGS. 2A and 2B. Flow rate is such that fluidic force 104and magnetic force 106 detach, on average over the cells in the cellsuspension, at least one paramagnetic particle 102 from cell 100 whilecell 100 passes through chamber 50. Flow rate is also such that thecells are not lysed by fluidic forces.

FIG. 8A shows module 8 of FIG. 2B when paramagnetic particle-bound cells100 a and 100 b have recently entered chamber 50. Cell 100 a has oneparamagnetic particle 102, while cell 100 b has two paramagneticparticles 102. In FIG. 8B, one paramagnetic particle 102 each hasdetached from both cell 100 a and cell 100 b and has come to rest onwall 52, while cells 100 a and 100 b continue to pass through chamber50. Cell 100 a no longer has any paramagnetic particles 102, while cell100 b retains one paramagnetic particle 102. Cell 100 b may be passedthrough module 8 a second time to remove this second paramagneticparticle 102. Alternatively, module 8 may be configured such that cell100 b remains in chamber 50, similar to the paramagnetic particle-boundcell in FIG. 7, while cell 100 a passes out of chamber 50.

This flow-through debeading process may remove at least 99% ofparamagnetic particles from cells.

When a system 2 containing a recirculation loop 14 from magnetic outputmodule 12 was used to debead CLT0119 T cells, cells in the magneticoutput were resuspended in buffer from buffer module 6 and passedthrough module 8 a second time and then again a third time. A comparisonof the results of this process to the results of a conventionalstop-flow process are provided in FIG. 9. FIG. 9 presents end-to-endyields, which are the ratios of the number of cells in the final productto the number of cells that entered the debeading system.

The flow-through magnetic separation/debeading module 8 shown in FIG. 3may be used to debead cells in a manner similar to module 8 as shown inFIGS. 2A and 2B. Fluid introduced through ports 72 supplies a force tocells 100 sufficient to dislodge them from membrane 70. Because velocityof the suspension fluid approached zero near membrane 70, these cellsmight otherwise not be separated or debeaded or may be lost.

This flow-through debeading process may also remove at least 99% ofparamagnetic particles from the cells.

Flow-Through Zero Gradient Filter Process

A flow-through separation process may be conducted using flow-throughmagnetic separation/debeading module 8 in a configuration as shown inFIGS. 4A and 4B. Flow rate is such that fluidic force 104 and magneticforce 106 allow paramagnetic particle 102 to remain bound to cell 100.Flow rate is also such that cell 100 does not stop in chamber 50 andalso is not lysed by fluidic forces. Various configurations are shown inFIGS. 10-13 and may be modified, for example to adjust the number andproportional size of inlets 62 and outlets 64, for use with differentzero gradient filter processes. For instance, the same effects achieveby having inlets 62 and outlets 64 of different sizes can also be theachieved by providing different flow rates, typically controlled bypumps, through same-size inlets and outlets.

FIG. 10 presents a basic description of how paramagnetic particle-boundcells 100 flow-through module 8 in a zero gradient configuration. Asillustrated in FIG. 10A, cells 100 after entering module 8 (left figure)are pulled in the x-direction to zero gradient line 58 by the time itthey are nearly exiting module 8 (right figure). As illustrated in FIGS.10A and 10B, cells 100 are not subject to magnetic force effects in they-direction or the z-direction when entering module 8 (left figure) oreven when close to exiting (right figure).

FIG. 11 shows a zero-gradient module 8 with magnets 54 oriented as shownin FIG. 4A. In this module, fluid enters via inlet 62. Cells 100 withmagnetic particles 102 follow zero gradient line 58 and are directed tomagnetic outlet 64 b. Unbound cells 110 are unaffected by zero gradientline 58 and flow to non-magnetic outlets 64 a and 64 c. Some unboundcells 110 will also enter magnetic outlet 64 b in this configuration.

FIG. 12 shows cells moving through module 8 of FIG. 4C. Cells 100 withparamagnetic particles 102 follow zero gradient line 58 and are directedto magnetic outlet 64 a. Unbound cells 110 are unaffected by zerogradient line 58 and flow to non-magnetic outlet 64 b. Thus, zerogradient line 58 acts as a magnetic filter while allowing all cells tocontinue to move through chamber 50 without being pushed towards anywall 52. As illustrated non-magnetic inlet 62 a is larger than magneticinlet 62 b and non-magnetic outlet 64 b is larger than magnetic outlet64 a. If fluid flow in module 8 is laminar, fluid from non-magneticinlet 62 a crosses over to non-magnetic outlet 64 b, preventing anyunbound cells 110 from entering magnetic outlet 64 a. This method mayalso be used with turbulent flow, but with less efficiency due to lossof unbound cells to the magnetic outlet 64 a.

This flow-through separation process may remove at least 80%, at least90%, at least 95%, or at least 99% of paramagnetic particle-bound cells100 from the cell suspension prior to its entry into non-magnetic outputmodule 10. Multiple passes of either the non-magnetic output or themagnetic output through flow-through magnetic separation/debeadingmodule 8 may result in a paramagnetic particle-bound cell product withno more than 1% unbound cells 11 and containing at least 99% of theparamagnetic particle-bound cells 100 found in the cell suspension priorto the flow-through separation process, an unbound cell product with nomore than 1% paramagnetic particle-bound cells 100 and containing atleast 99% of the unbound cells 110 found in the cell suspension prior tothe flow-through process, or both.

Flow-Through Zero Gradient Paramagnetic Particle Separation Process

A flow-through paramagnetic particle separation process may also beconducted using flow-through magnetic separation/debeading module 8 in aconfiguration as shown in FIG. 4C. FIG. 13 shows debeaded, unbound cells110 and paramagnetic particles 102 moving through module 8. Cells 110were previously debeaded, for instance by spinning membrane debeadingmodule 18, a non-spinning membrane debeading module, or the same or aseparate module 8 in a debeading configuration. Alternatively, cells 110may have already been in the presence of, but not bound to paramagneticparticles 102. Paramagnetic particles 102 follow zero gradient line 58and are directed to magnetic outlet 64 a. Debeaded, unbound cells 110are unaffected by zero gradient line 58 and flow to non-magnetic outlet64 b. Thus, zero gradient line 58 acts as a magnetic filter whileallowing cells 110 to continue to move through chamber 50 without beingpushed towards any wall 52.

This flow-through paramagnetic particle separation process may remove atleast 80%, at least 90%, at least 95%, or at least 99% of beads from thecell suspension.

This process, in conjunction with removing the paramagnetic particles,may remove an unwanted constituent of the cell suspension as well.

Although the cell separation process and the particle separation processare described separately above, they may both occur simultaneously inthe same module or system. For example, a separation module willtypically remove both paramagnetic particle-bound cells and freeparamagnetic particles from the cell suspension.

Spinning Membrane Debeading Processes

In a flow-through process using system 2 of FIG. 1F, separatedparamagnetic particle-bound cells 100 are directed into spinningmembrane module 18 as shown in FIG. 14 via sample inlet 80. Withindebeading chamber 82, spinning membrane 86 generates recirculatingTaylor-Couette flows in the cell suspension which cause fluidic forcesin addition to the fluidic forces generated by flow through chamber 82from inlet 80 to outlets 20 and 22. As a result, although paramagneticparticles 102 still experience a fluidic force and a magnetic force, therelationship of these forces and how they cause debeading is difficultto model. However, fluidic forces are affected by at least the size andspin rate of spinning membrane 80, the cell suspension flow rate throughchamber 82, and the viscosity of the suspension fluid. Magnetic force isaffected by the nature of paramagnetic particles 102, the nature ofmagnets 88, and the design of module 18, particularly distance betweenthe cells 100 and magnets 88. Fluidic forces are typically not so highas to lyse the cells. The Taylor-Couette flows are sufficient to keepthe cells away from and out of contact with wall 84 and spinningmembrane 86.

Paramagnetic particles 102 that are removed from cells 100 migrate towall 84, and particularly to zero gradient lines or bands along wall 84.Any non-paramagnetic particles 120 are also removed from cells 100 byfluidic forces alone. Non-paramagnetic particles 120 pass through poresin spinning membrane 86 and then exit chamber 82 via waste outlet module20. Any chemical agent 122 added from optional reagent chamber 24 alsopasses through the pores in spinning membrane 86 and exits chamber 82via waste outlet module 20. This limits exposure of cells 100 tochemical agent 120. Debeaded, unbound cells 110 exit chamber 82 via celloutlet module 22.

Paramagnetic particles 102 may be removed from wall 84 periodically, forinstance by stopping cell suspension flow through chamber 82, movingmagnets 88 to a position distant from wall 84, then flowing bufferthrough chamber 82.

Some paramagnetic particles 102 may also pass through spinning membrane80 and be removed. If magnets 88 are absent or sufficiently distant fromchamber 82, all paramagnetic particle removal may be accomplished byspinning membrane 80.

This flow-through debeading process may also remove at least 80%, atleast 90%, at least 95%, or 99% of paramagnetic particles from thecells, at least 80%, at least 90%, at least 95%, or at least 99% of allnon-paramagnetic particles from the cells, or both.

The spinning membrane may also be used to separate paramagneticparticles 102 from unbound cells 102.

Spinning membrane 86 may have a pore size small enough to exclude allcells in the cell suspension.

Spinning membrane module 18 may also be used to remove unwantedconstituents from the cell suspension. These constituents may simply befiltered by spinning membrane 88, or they may interact with the coatingof paramagnetic particles 102, non-paramagnetic particles 120, or both,and be removed with the particles. Debeading and unwanted constituentremoval may occur separately or simultaneously.

Other Debeading and Paramagnetic Particle Separation Processes

System 2, due to it modular design, is also compatible with otherdebeading and paramagnetic particle separation processes. One need onlyinsert an appropriate additional module 16. For instance, columns,including magnetic columns and physical separation methods are oftenused to debead cells and may be included as an additional module 16.

Other Incorporated Processes

System 2, due to its modular design, is compatible with otherincorporated processes. These processes may occur in at least oneadditional module 16. For instance, a module may be used to wash cells.A module may also be used to concentrate cells. A module may be used toexchange the media in which cells are located. One module may be usedfor more than one of these steps.

Multiple-Module Flow-Through Process

System 2 as shown in FIG. 1G may be used in a multiple-moduleflow-through process. Optionally, buffer from buffer module 6 a may beflowed through flow-through magnetic separation/debeading module 8 a andoptionally also one or more of modules 8 b, 18 a, and 18 b. A cellsuspension containing desirable, paramagnetic particle andnon-paramagnetic particle-bound cells and undesirable unbound cells isflowed through module 8 a, which is configured as shown in FIG. 4 forseparation, but alternatively may be configured as shown in FIGS. 2A and2B for separation. Unbound cells are directed to non-magnetic outputmodule 10 a as waste. Paramagnetic particle-bound cells are directed viamagnetic output module 12 a to flow-through magneticseparation/debeading module 8 b. Module 8 b is configured as shown inFIGS. 2A and 2B for debeading. Paramagnetic particle-bound cells areflowed through return loop 14 a at least once prior to entering magneticoutput module 12 b as waste. Debeaded, unbound cells are sent vianon-magnetic output module 10 b to spinning membrane debeading module 18a. Any remaining paramagnetic particles and non-paramagnetic particlesare removed, with non-paramagnetic particles flowing into waste outputmodule 20 a, while unbound paramagnetic particles remain in module 18and unbound cells and cells with paramagnetic particles,non-paramagnetic particles, or both flow into cell output module 22 a.Cell output module 22 a leads to a second spinning membrane module 18 b.A chemical agent able to facilitate removal of either the paramagneticparticle or the non-paramagnetic particle, or both, from the cells isadded from reagent module 24. Non-paramagnetic particles and thechemical agent flow into waste module 20 b. Paramagnetic particlesremain in module 18. Unbound cells and cells with either paramagneticparticles, non-paramagnetic particles, or both flow into return loop 14b at least once prior to being sent to cell output module 22 b as thefinal cell product of the flow-through process.

Clinical Applications

All of the processes herein may be conducted according to clinical goodmanufacturing practice (cGMP) standards.

The processes may be used for cell purification, enrichment, harvesting,washing, concentration or for cell media exchange, particularly duringthe collection of raw, starting materials (particularly cells) at thestart of the manufacturing process, as well as during the manufacturingprocess for the selection or expansion of cells for cell therapy.

The cells may include any plurality of cells. The cells may be of thesame cell type, or mixed cell types. In addition, the cells may be fromone donor, such as an autologous donor or a single allogenic donor forcell therapy. The cells may be obtained from patients by, for example,leukapheresis or apheresis. The cells may include T cells, for examplemay include a population that has greater than 50% T cells, greater than60% T cells, greater than 70% T cells, greater than 80% T cells, or 90%T cells.

Selection processes may be particularly useful in selecting cells priorto culture and expansion. For instance, paramagnetic particles coatedwith anti-CD3 and/or anti CD28 may be used to select T cells forexpansion or for introduction of a nucleic acid encoding a chimericantigen receptor (CAR) or other protein. Such a process is used toproduce CTL019 T cells for treatment of acute lymphoblastic leukemia(ALL).

The debeading processes and modules disclosed herein may be particularlyuseful in the manufacture of cells for cell therapy, for example inpurifying cells prior to, or after, culture and expansion. For instance,paramagnetic particles coated with anti-CD3 and/or anti CD28 antibodiesmay be used to selectively expand T cells, for example T cells that are,or will be, modified by introduction of a nucleic acid encoding achimeric antigen receptor (CAR) or other protein, such that the CAR isexpressed by the T cells. During the manufacture of such T cells, thedebeading processes or modules may be used to separate T cells from theparamagnetic particles. Such a debeading process or module is used toproduce, for example, CTL019 T cells for treatment of acutelymphoblastic leukemia (ALL).

In one such process, illustrated here by way of example, cells, forexample, T cells, are collected from a donor (for example, a patient tobe treated with an autologous chimeric antigen receptor T cell product)via apheresis (e.g., leukapheresis). Collected cells may then beoptionally purified, for example, by an elutriation step. Paramagneticparticles, for example, anti-CD3/anti-CD28-coated paramagneticparticles, may then be added to the cell population, to expand the Tcells. The process may also include a transduction step, wherein nucleicacid encoding one or more desired proteins, for example, a CAR, forexample a CAR targeting CD19, is introduced into the cell. The nucleicacid may be introduced in a lentiviral vector. The cells, e.g., thelentivirally transduced cells, may then be expanded for a period ofdays, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days, forexample in the presence of a suitable medium. After expansion, thedebeading processes/modules disclosed herein may be used to separate thedesired T cells from the paramagnetic particles. The process may includeone or more debeading steps according to the processes of the presentdisclosure. The debeaded cells may then be formulated for administrationto the patient. Examples of CAR T cells and their manufacture arefurther described, for example, in WO2012/079000, which is incorporatedherein by reference in its entirety. The systems and methods of thepresent disclosure may be used for any cellseparation/purification/debeading processes described in or associatedwith WO2012/079000.

The systems and methods herein may similarly benefit other cell therapyproducts by wasting fewer desirable cells, causing less cell trauma, andmore reliably removing magnetic and any non-paramagnetic particles fromcells with less or no exposure to chemical agents, as compared toconventional systems and methods.

Example

The following example is provided for illustrative purposes only and isnot intended to encompass the entire invention. Aspects of this examplemay be combined with other aspects of the invention described above.

In this example, T cells were expanded over a 9-day period in culture,then harvested and debeaded using a non flow-through debeading processaccording to prior procedures or a flow-through debeading processaccording to the present disclosure. The samples had betweenapproximately 1e8 nucleated viable cells and approximately 3e10nucleated viable cells. The paramagnetic particle to nucleated viablecell ratio was between approximately 3:1 and 1:3, with samples havinglower total nucleated viable cells exhibiting higher paramagneticparticle to nucleated viable cell ratios. The paramagnetic particle tocell ratio was a significant factor in cell recovery. A higherparamagnetic particle to nucleated viable cell ratio increased thechances that a nucleated viable cell is bound to a paramagnetic particleand is lost during the paramagnetic particle removal process.

In the non flow-through debeading process, the sample was collected in aone liter platelet bag (Terumo Medical Corp., Somerset, N.J.) andstatically placed on top a flat-bed magnetic plate (DYNAMAG™ CTS™,Thermo Fisher Scientific, Waltham, Mass.) of 5 minutes at zero degrees,followed by one minute at a 30 degree inclination. Next, the liquid inthe bag, which contained the non-magnetic fraction, was diverted fromthe bag to form the final product. The magnetic fraction remained insidethe bag as waste.

In the flow-through debeading process, the sample was continuouslyflowed through a CSD400Y9 CRYOSTORE™ Conical Bag (OriGen Biomedical,Austin, Tex.) placed on top a flat-bed magnetic plate (DYNAMAG™ CTS™,Thermo Fisher Scientific, Waltham, Mass.). Due to continuous flowthrough the bag and over the magnet, the sample was dynamicallydebeaded, with paramagnetic particles being stripped off the viablenucleated cells as they moved through the bag. The liquid afterpassaging through the bag for some time formed the final product. Themagnetic fraction remained inside the bag as waste. Few viable nucleatedcells were trapped in the magnetic faction. This is in contrast to thenon flow-through debeading process, which viable nucleated cells boundto paramagnetic particles were attracted to the magnet and lost in thewaste.

Metadata analysis of thirty-eight non flow-through debeading processruns and thirty-six flow-through debeading process runs showedsignificant increases in recovery of viable nucleated cells when theflow-through debeading process was used. FIG. 15. The increase in viablenucleated cell recovery was particularly significant at lower numbers ofcells in the sample (such as less than 1.6e9 total viable nucleatedcells). At such total viable nucleated cell numbers, the flow-throughdebeading process exhibited a 76% average recovery as compared to only34% average recovery for the non flow-through debeading process. FIG.16. A 10-20% increase in recovery was also seen with higher numbers ofcells in the sample.

This difference in recovery is due to the ability of the flow-throughdebeading process to dynamically remove paramagnetic particles from theviable nucleated cells, so that these cells are not lost even if theywere initially bound to paramagnetic particles prior to harvest.

Although only exemplary embodiments of the disclosure are specificallydescribed above, it will be appreciated that modifications andvariations of these examples are possible without departing from thespirit and intended scope of the disclosure. For example, the magneticmodules and systems containing them may be arranged and used in avariety of configurations in addition to those described. In addition,the systems and methods may include additional components and steps notspecifically described herein. For instance, methods may includepriming, where a fluid is first introduced into a component to removebubbles and reduce resistance to cell suspension or buffer movement.Furthermore, embodiments may include only a portion of the systemsdescribed herein for use with the methods described herein. For example,embodiments may relate to disposable modules, hoses, etc. usable withinnon-disposable equipment to form a complete system able to separate ordebead cells to produce a cell product.

1. A cell processing system comprising: at least one cell suspensionmodule; at least one buffer module; at least one flow-through magneticseparation/debeading module; at least one non-magnetic output module;and at least one magnetic output module.
 2. The cell processing systemof claim 1, further comprising at least one return loop returningupstream of at least one flow-through magnetic separation/debeadingmodule.
 3. The cell processing system of claim 1, comprising at leasttwo flow-through magnetic separation/debeading modules in parallel. 4.The cell processing system of claim 1, comprising at least twoflow-through magnetic separation/debeading modules in series.
 5. Thecell processing system of claim 1, further comprising at least oneadditional module.
 6. The cell processing system of claim 5, wherein theat least one additional module comprises at least one spinning membranedebeading module.
 7. The cell processing system of claim 6, comprisingat least two spinning membrane debeading modules in parallel.
 8. Thecell processing system of claim 6, comprising at least two spinningmembrane debeading modules in series.
 9. The cell processing system ofclaim 5, wherein the at least one additional module comprises at leastone physical separation module.
 10. The cell processing system of claim9, wherein the at least one additional module comprises at least onemagnetic column module.
 11. The cell processing system of claim 5,wherein the at least one additional module comprises at least one mediaexchange module.
 12. The cell processing system of claim 5, wherein theat least one additional module comprises at least one cell concentrationmodule.
 13. The cell processing system of claim 5, wherein the at leastone additional module comprises at least one cell washing module. 14.The cell processing system of claim 1, wherein the flow-through magneticseparation/debeading module comprises: a chamber defined by walls andhaving an x-direction, a y-direction, and a z-direction; an inlet and anoutlet arranged on opposite ends of the chamber in the y-direction; andat least two magnets adjacent or proximate a wall of the chamber andarranged to establish a zero gradient line within the chamber betweenthe inlet and the outlet.
 15. The cell processing system of claim 6,wherein the spinning membrane debeading module comprises: a debeadingchamber define partially by a cylindrical side-wall; a porous spinningmembrane having an interior and oriented co-axially with the cylindricalside-wall; a sample inlet; a waste output module connected to theinterior of the spinning membrane; and a cell output module connected tothe debeading chamber.
 16. The cell processing system of claim 15,wherein the spinning membrane debeading module further comprises atleast one magnet adjacent or proximate to the cylindrical side-wall. 17.A flow-through magnetic separation/debeading module comprising: achamber defined by walls and having an x-direction, a y-direction, and az-direction; an inlet and an outlet arranged on opposite ends of thechamber in the y-direction; and at least two magnets adjacent orproximate a wall of the chamber and arranged to establish a zerogradient line within the chamber between the inlet and the outlet. 18.The module of claim 17, comprising at least two inlets and at least twooutlets.
 19. The module of claim 17, further comprising at least threemagnets adjacent or proximate a wall of the chamber and arranged toestablish at least two zero gradient lines within the chamber betweenthe inlet and the outlet.
 20. The module of claim 17, further comprisingat least four magnets arranged in two arrays on opposite sides of thechamber in the z-direction.
 21. The module of claim 18, furthercomprising at least four magnets arranged in two arrays on opposite sideof the chamber in the z-direction and cross-oriented in the x-y planefrom near one inlet to near one outlet on the opposite side of thechamber in the z-direction.
 22. The module of claim 17, furthercomprising: sub-membrane injection ports adjacent a wall of the chamberalso adjacent at least two magnets; and a membrane adjacent thesub-membrane.
 23. A spinning membrane debeading module comprising: adebeading chamber define partially by a cylindrical side-wall; a porousspinning membrane having an interior and oriented co-axially with thecylindrical side-wall; a sample inlet; a waste output module connectedto the interior of the spinning membrane; a cell output module connectedto the debeading chamber; and at least one magnet adjacent or proximateto the cylindrical side-wall.
 24. The spinning membrane debeading moduleof claim 23, further comprising a reagent module.
 25. The spinningmembrane debeading module of claim 23, wherein the porous spinningmembrane has a pore size greater than that of a particle to be debeadedand less than that of a cell to be debeaded.
 26. A method offlow-through cell processing comprising flowing a cell suspensioncomprising paramagnetic particle-bound cells through a flow-throughmagnetic separation/debeading module to produce an unbound cell product,wherein the paramagnetic particle-bound cells continue to move in theflow-through magnetic separation/debeading module through the flowingstep, and wherein the flow-through magnetic separation/debeading modulecomprises: a flow chamber defined by walls through which the cellsuspension flows; and at least two magnets arranged adjacent orproximate at least one wall.
 27. The method of claim 26, wherein thecell suspension is flowed laminarly through the flow-through magneticseparation/debeading module.
 28. The method of claim 26, wherein thecell suspension further comprises unbound cells and flowing the cellsuspension through the flow-through magnetic separation/debeading moduleseparates the paramagnetic particle-bound cells and the unbound cells.29. The method of claim 28, wherein the cell suspension furthercomprises free paramagnetic particles and flowing the cell suspensionthrough the flow-through magnetic separation/debeading module separatesthe free paramagnetic particles and the unbound cells.
 30. The method ofclaim 28, further comprising flowing the separated unbound cells throughthe flow-through magnetic separation/debeading module a second orsubsequent time using a return loop.
 31. The method of claim 28, furthercomprising flowing the separated paramagnetic particle-bound cellsthrough the flow-through magnetic separation/debeading module a secondor subsequent time using a return loop.
 32. The method of claim 31,further comprising debeading the paramagnetic particle-bound cells inthe flow-through magnetic separation/debeading module during the secondor subsequent time to produce paramagnetic particles and debeaded,unbound cells.
 33. The method of claim 32, further comprising flowingthe produced paramagnetic particles and debeaded, unbound cells throughthe flow-through magnetic separation/debeading module a third orsubsequent time to separate the paramagnetic particles and the debeaded,unbound cells.
 34. The method of claim 26, wherein the magnets areoriented to establish one zero gradient line that crosses the directionof flow, such that paramagnetic-particle bound cells are pulled to thezero gradient line in one direction only, but are not affected bymagnetic forces of the two magnets in two other directions.
 35. Themethod of claim 26, wherein the chamber further comprises: a magneticinlet through which any paramagnetic particles enter the flow chamber; anon-magnetic inlet; a magnetic outlet opposite the non-magnetic inlet;and a non-magnetic outlet opposite the magnetic inlet, wherein the zerogradient line directs all paramagnetic particles and any bound cells tothe magnetic outlet.
 36. The method of claim 35, wherein the cellsuspension further comprises unbound cells and wherein non-magneticinlet is larger than the magnetic inlet and the non-magnetic outlet islarger than magnetic outlet, wherein fluid flowing from the non-magneticinlet crosses over to the non-magnetic outlet, preventing any unboundcells from flowing into the magnetic outlet.
 37. The method of claim 35,wherein the cell suspension further comprises unbound cells, wherein thenon-magnetic inlet and magnetic inlet are substantially the same size orthe non-magnetic out and magnetic outlet are substantially the samesize, or both, and wherein respective flow rates of the fluid enter theinlets, the respective flow rates of the fluid exiting the outlets, orboth are adjusted such that fluid flowing from the non-magnetic inletcrosses over to the non-magnetic outlet, preventing any unbound cellsfrom flowing into the magnetic outlet.
 38. The method of claim 26,further comprising flowing the paramagnetic particle-bound cells througha spinning membrane debeading module to produce the unbound cellproduct, wherein the spinning membrane debeading module comprises: acylindrical debeading chamber through which the paramagneticparticle-bound cells flow, the chamber defined in part by a cylindricalside-wall and containing a co-axial spinning membrane; and at least onemagnet arranged adjacent or proximate the cylindrical side-wall toestablish at least one zero gradient line within the cylindricaldebeading chamber.
 39. The method of claim 26, further comprisingflowing the paramagnetic particle-bound cells through a magnetic columnmodule to produce the unbound cell product.
 40. The method of claim 26,further comprising flowing the paramagnetic particle-bound cells or theunbound cell product through a cell washing module.
 41. The method ofclaim 26, further comprising flowing the paramagnetic particle-boundcells or the unbound cell product through a media exchange module. 42.The method of claim 26, further comprising flowing the paramagneticparticle-bound cells or the unbound cell product through a cellconcentration module.
 43. A method of manufacturing a cell therapycomposition, said method comprising: contacting a cell population withparamagnetic particles coated with one or more agents which assist inexpanding one or more cell types within the cell population; introducingnucleic acid into cells within the cell population; expanding cellswithin the cell population; debeading the cell population according tothe method of any of claims 26 to 42, or using the system of any ofclaims 1 to 16 or the module of any of claims 17-25; and formulating thecell population for cell therapy.
 44. The method of claim 43, whereinthe one or more agents which assist in expanding one or more cell typescomprises anti-CD3 antibody or antigen binding fragment thereof,anti-CD28 antibody or antigen binding fragment thereof, and combinationsthereof.
 45. The method of any of claims 43 to 44, wherein the nucleicacid is introduced by lentivirus or mRNA transduction.
 46. The method ofany of claims 43 to 45, wherein the cell therapy is a chimeric antigenreceptor T cell therapy.
 47. The method of claim 46, wherein the celltherapy is an anti-CD19 chimeric antigen receptor T cell therapy.